Substituted 7-and/or 9-amino-6-methylenetetracyclines



United States Patent 3,341,586 SUBSTITUTED 7- AND/0R 9-AMINO-6- METHYLENETETRACYCLINES Panayota Bitha, New York, Joseph John Hlavka, Tuxedo, and Michael Joseph Martel], Jr., Pearl River, N.Y., assignors to American Cyanamid Company, Stamford, Conn., a corporation of Maine No Drawing. Filed Dec. 1, 1966, Ser. No. 598,232

10 Claims. (Cl. 260-559) ABSTRACT OF THE DISCLOSURE This disclosure describes compounds of the class of 7- and/or 9-mono(lower alkyl)aminoor -di(lower alkyl) amino-G-methylenetetracyclines useful as antibacterial agents.

This application is a continuation-in-part of our copending application Serial No. 548,076, filed May 6, 1966 now abandoned.

This invention relates to new compounds of the tetracycline family and, more particularly, is concerned with novel substituted 7- and/or 9-amino-6-methylenetetracyclines which may be represented by the following general formula:

wherein R is hydrogen, a-hydroxy or fi-hydroxy and R and R are hydrogen, mono(lower alky1)amino or di (lower alky1)amino with the proviso that R and R cannot both be hydrogen. Typical compounds represented by the above general formula are, for example,

These new tetracycline derivatives may be prepared by a reductive alkylation process comprising interacting a compound of the following general formula:

wherein R is hydrogen, a-hydrOXy or p-hydroxy and Y and Z are hydrogen, amino, a substituent reducible to amino, mono(lower alkyl)amino or a substituent reducible to mono(lower alkyl)amino with the proviso that Y pound of the general formula:

wherein R is hydrogen or lower alkyl and R is hydrogen or lower alkyl; in the presence of a reducing agent.

It is to be understood that when the term lower alkyl is used throughout this specification, it is alkyl groups having up to about 6 carbon atoms. Accordingly, aldehydes and ketones useful in car rying out this reductive alkylation include, for example, formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, isobutyraldehyde, acetone, methyl ethyl ketone, diethyl ketone, etc.

In the second general formula set forth above, the substituents Y and Z are defined as hydrogen, amino, a substituent reducible to amino, mono(lower alkyl)amino or a substituent reducible Suitable substituents reducible to amino may be, for example, nitro, nitroso, benzeneazo, substituted-benzeneazo, etc. Suitable substituted-benzeneazo groups may be, for

such products may be prepared in either of two ways. In the first situation, Y and/or Z may be amino or a substituent reducible to amino and reductive dialkylation on an unsubstituted amino group occurs, whereby a disubstituted amino group is obtained. In the second situation, Y and/0r Z may be mono(lower alkyl)amino or a substituent reducible to mono (lower aIkyDamino and reductive monoalkylation on a monoalkyl-substituted amino for example, methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylarnino, t-butylarnino, etc. Suitable substituents reducible to mono (lower alkyl) amino may be, for example, formylamino, acetylamino, N-(lower alkyl)hydroxylamino, and the like. Specific starting materials operable in this process include,

7-nitro-6-methylenetetracycline, 7-amino-6-methylenetetracycline, 7-nitroso-6-methylenetetracycline, 9-nitro-6-methylenetetracycline, 9-amino-6-methylenetetracycline, 9-phenylazo-6-methylenetetracycline, 7-nitro-5-ot-hydroxy-6-methylenetetracycline, 7-amino-5-a-hydroxy-G-methylenetetracycline, 7-p-sulfophenylaZo-5-a-hydroXy-6-methylenetetracycline, 9-amino-5-a-hydroxy-6-methylenetetracycline, 9-nitroso-5-a-hydroxy-6-methylenetetracycline, 9-phenylazo-5-a-hydroxy-6-methylenetetracycline, 7-nitro-5-B-hydroxy-6-methylenetetracycline, 7-amino-5-fl-hydroxy-6-methylenetetracycline, 7-nitroso-5-,8-hydroxy-6-methylenetetracycline, 7-p-nitrophenylazo-S-fi-hydroxy-G-methylenetetracycline, 9-nitro-5-;8-hydroXy-6-methylenetetracycline, 7,9-dinitro-G-methyIenetetracycline, 7,9-diamino-5-a-hydroxy-6-methylenetetracycline, and 7,9-di (phenylazo) -5- 3-hydroxy-6-methylenetetracycline.

The 7- and/or 9-nitro-fi-methylenetetracycline starting at a temperaure of from about for a period of time of from about several minutes to about several hours or more. If the mononitro derivative is desired then one molar equivalent of nitronium tetrafluoroborate is employed, whereas if a dinitro derivative is desired then two molar equivalents of nitronium tetrafluoroborate are employed. The 7- and/or 9-nitroso-6-methylenetetracycline starting materials may be readily prepared by treating an appropriate G-methylenetetracycline with nitrous acid in acidic aqueous medium. The 7- and/or 9-benzeneazo or substituted-benzeneazo 6-methylenetetracycline starting materials may be readily prepared by coupling an appropriate 6-methylenetetracycline with benzene diazonium halide or a substituted-benzene diazonium halide under conditions well known in the art.

The 7- and/or 9-amino'6-methylenetetracycline starting materials for this process may be prepared by the catalytic reduction of the corresponding 7- and/ or 9-nitro, nitroso, phenylazo or substituted-phenylazo 6-methylenetetracyclines. The 7- and/ or 9-amino-6-rnethylenetetracycline starting materials may also be prepared by the catalytic reduction of the corresponding 11a-chloro-7- and/ or 9-nitro, nitroso, phenylazo or substitutedphenylazo 6-methylenetetracyclines. In this catalytic reduction, the lla-chloro group is concurrently removed while the 7- and/ or 9-substituent is reduced to amino. These 11achloro-7- and/ or 9-substituted-6-methylenetetracycline intermediates may also be employed directly in the reductive alkylation step whereupon the lla-chloro group is concurrently removed. The 11a-chloro-7- and/ or 9-nitro, nitroso, phenylazo or substituted-phenylazo 6-methylenetetracycline intermediates may be readily prepared by nitrating, nitrosating, or coupling with benzene diazonium halide or a substituted-benzene diazonium halide, an appropriate lla-chloro-6-methylenetetracycline under the same general conditions as are employed with the 11aunblocked compounds.

The reductive alkylation process may be accomplished by either chemical or catalytic reduction using procedures well-known to those in the art. Catalytic reduction, which is especially suited for the reductive alkylation of the tetracycline starting compounds set forth above, may be accomplished in a solvent for the tetracycline starting compound in the presence of a carbonyl compound and a metal catalyst and hydrogen gas at pressures from atmospheric to super-atmospheric. Ordinarily, the reductive alkylation is conveniently carried out at hydrogen pressures of from about one to about four atmospheres. Temperature does not appear to be critical in the catalytic hydrogenation. Temperatures of from C. to 50 C., and usually room temperature, are preferred since they generally give best results. The metal catalyst may be of the base metal type, such as nickel or copper chromite, or it maybe of the noble metal type, such as finely divided platinum, palladium or rhodium. The noble metal catalysts are advantageously employed on a carrier such as finely divided alumina, activated charcoal, diatomaceous earth, etc., in which form they are commonly available. The hydrogenation is carried out until the desired amount of hydrogen gas is absorbed at which point the hydrogenation is stopped. The solvents selected for the catalytic reduction should be reaction-inert, that is, they should not be capable of reacting with the starting materials, product, or hydrogen under the conditions of the reaction. A variety of solvents may be used for this purpose and minimum laboratory experimentation will permit the selection of a suitable solvent for any specific tetracycline starting compound. Generally, the catalytic reductive alkylation may be carried out in solvents such as water; lower alkanols, e.g. methanol, ethanol; lower alkoxy lower alkanols, e.g. Z-methoxyethanol, 2-ethoxyethanol; tetrahydrofuran; dioxane; dimethylformamide;

etc.

A variety of chemical reducing agents may be used in the reductive alkylation process. These include reduction with active metals in mineral acids, e.g. zinc, tin, or iron in hydrochloric acid; reduction with metal couples such as the copper-zinc couple, the tin-mercury couple, aluminum amalgam or magnesium amalgam; reduction with lithium aluminum hydride or sodium borohydride; and reduction with formic acid. Of these, reduction with zinc and hydrochloric acid and reduction with formic acid are preferred. When aqueous systems are used in the aforementioned chemical reductive alkylations, it is at times desirable to utilize a water-miscible organic solvent, particularly when the tetracycline starting compound is of limited solubility in the reaction mixture. The water-miscible solvent does not alter the course of the reduction but merely provides for more efficient reduction, e.g. a shorter reaction time by providing more intimate contact of the reagents. A large number of such solvents are available for this purpose and include, among other, dimethylformamide, dimethoxyethane, methanol, ethanol, dioxane, tetrahydrofuran, and the like.

The novel products of the present invention are obtained from the reductive alkylation reaction mixtures by standard procedures. For example,.the products may be isolated from the catalytic hydrogenation reaction mixtures, after filtration of the catalyst, by precipitation with a solvent such as ether or hexane or by concentration, usually under reduced pressure, or by a combination of these. Workup of the chemical reductive alkylation reaction mixtures to obtain the desired products may also be accomplished by known procedures such as precipitation, concentration, solvent extraction, or combinations of these procedures. by any of the generally known methods for purification of tetracycline compounds. These include recrystallization from various solvents and mixed solvent systems, chromatographic techniques, and counter current distribution,

all of Which are usually employed for this purpose.

The new tetracyclines of this invention are biologically active and have the broad-spectrum antibacterial activity of the previously known tetracyclines. The antibacterial spectrum of certain of these compounds, representing the amount required to inhibit the growth of various typical bacteria, was determined in a standard manner by the agar dilution streak plate technique which is commonly used in testing new antibiotics. The following table summarizes the in vitro activity of 7-dimethylamino-5-ahydroxy-6-methylenetetracycline (l) as compared with 7-amino-5-u-hydroxy-6-methylenetetracycline (2) against a variety of disease-causing microorganisms.

Minimal inhibitory cone. (meg lmi) Organism.

M ycobactertum smcgmatts ATCC 607.. 3. 1 3.1 Staphylococcus aurcus ATCC 6538? 12. 5 31 Staphylococcus aurzus ATOC 13709- 6. 2 12. 5 Sorciua lutea ATCC 9341 6.2 12. 5 Streptococcus faecatis ATC O 8043 6. 2 31 Streptococcus pyogeues (3-203 3.1 6.2 Bacillus subttlis ATCC 6633 3. 1 12. 5 Coryuebactcrtum xerosts NRLL B-1397 3. 1 6.2 Bacillus ccreus ATCC 10702 3.1 6. 2 Klebstella pucumontue ATCC 10031 6. 2 31 The present new tetracycline compounds may be formulated into various compositions analogous to the parent tetracyclines from which they are derived. They are useful therapeutically in feeds or as growth stimulants, in veterinary practice and in agriculture.

For human therapy, the usual oral dosage of the present new compounds is from about 0.1 to about 2 g. per day for the average adult. T he product is formulated into capsules or tablets containing from 25 to 250 mg. of antibiotic on an activity basis. Suspensions or solutions in various vehicles are prepared using concentrations ranging from 5 to 125 mg./ml. For parenteral administration intramuscularly or intravenously, the daily dose is re duced to about 0.05 to 1.0 g. comprise solutions of the antibiotic at concentrations ranging from 50 to mg./ml. Intravenous administra- After isolation, the products may be purified Intramuscular formulations 'tion is by means of isotonic solutions having antibiotic concentration of about 10 mg./ml. Both types of parenteral products are conveniently distributed as solid compositions for reconstitution. These products may also be used for topical applications in the usual extending media. In all instances, of course, the attending physician will indicate the dosage to fit the needs of a particular patient.

The new tetracyclines of this invention are amphoteric compounds and hence acid-addition salts, that is both monoand di-salts, may be readily prepared. The preferred acids are the non-toxic pharmaceutically acceptable acids, e.g. the mineral acids such as hydrochloric, sulfuric, and the like although organic acids such as trichloroacetic may also be used. The acid-addition salts may be prepared by treating the new tetracyclines with approximately one or two equivalents of the chosen acid in a suitable solvent. These new tetracyclines also form typical Mannich derivatives with organic aldehydic compounds and nitrogen bases.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Preparation 7- and 9-nitro-6-methylene-5-ahydroxytetracycline A suspension of 6-methylene-5-a-hydroxytetracycline hydrochloride (1 mmole) in nitromethane (50 ml.) is prepared. The nitronium tetrafluoroborate (1.5 mmole) is added and the so-obtained solution is stirred at room temperature for two hours. The product is precipitated with ether, Washed well, and dried. The two isomers are separated by partition column chromatography.

EXAMPLE 2 Preparation of 7 -p-sal foph enylaz0-Ha-chl0r0-5-ahydroxy tetracycline The 11a-chloro-5-a-hydroxytetracycline (1.48 g.; 3.0 mmole) is suspended in water (60 ml.). The suspension is cooled to 05 C. in an ice bath. The pH is adjusted to 7.5 with 1 N NaOH and is then stirred at 05 C. for about 15 minutes. A solution of anhydrous sodium carbonate (174.9 mg; 1.65 mmole) in water (12 ml.) is prepared. The sulfanilic acid monohydrate (630 mg.; 3.3 mmole) is added and the mixture is heated to get the acid into the carbonate solution. It is cooled to 05 C. and then the solution of sodium nitrite (240 mg. 3.6 mmole) in water (3 ml.) is added. This mixture is poured into a beaker containing 3.3 g. of cracked ice and 0.69 ml. of conc. HCl (8.75 mmole). In a few minutes the white crystals of the diazonium salt precipitate out. The pH of the 11a-chloro-5-a-hydroxytetracycline suspension is checked and readjusted to 7.5. Then the suspension of the diazonium salt is added slowly While the pH is kept at 7.5 with 1 N NaOH. The so-obtained solution is stirred for 45 minutes in the ice bath. The ice is then removed and the solution is acidified to pH 1.5 with conc. HCl. The solid is centrifuged, transferred into a funnel, washed with water and dried.

EXAMPLE 3 Preparation of 7-p-sulfophenylazo-IIa-chl0r0-6- methylene-5-a-hydroxytetracycline The 7-p-sulfophenylazo 11a chloro-5-a-hydroxytetra cycline prepared in Example 2 (100 mg.) is dissolved in anhydrous hydrofluoric acid (5 ml.). The solution is stirred at 05 C. for 30 minutes. The acid is stripped 01f by passing nitrogen through the solution. The residue is received in acetone (2 ml.) and the product is precipitated with ether.

6 EXAMPLE 4 Preparation of 7-amz'no-6-methylene-5-ahydroxytetracycline EXAMPLE 5 Preparation of 7- and 9-nitro-6-methylene-1Iachlor0-5-a-hydroxytetracycline The 7- and 9-nitro-11a-chloro-5-a-hydroxytetracycline (100 mg.) is dissolved in hydrogen fluoride (5 ml.). The solution is stirred at 0-5 C. for 30 minutes. The acid is then stripped off by passing a stream of nitrogen through. The residue is received in ether and the solid filtered.

EXAMPLE 6 Preparation 0 7- and 9-nitro-6-methyIene-S-ahydroxytetracycline The mixture from the HF reaction of Example 5 mg.) is dissolved in water (20 ml.). A solution of sodium bisulfite (400 mg.) in water (5 ml.) is added and the pH is adjusted to 6.0. Solution is stirred at that pH for 30 minutes. It is then filtered and the filtrate is freeze-dried. The two isomers are separated by chromatography.

EXAMPLE 7 Preparation of 7- and 9-dimethylamino-6-methylene- 5-a-hydroxytetracycline The mixture of 7- and 9-amino-6-methylene-5-u-hydroxytetracyclines mg.) is dissolved in ethylene glycol monomethyl ether (10 ml.). There is added the conc. H SO (0.05 ml.), the 40% solution of formaldehyde in water (0.4 m1.) and the palladium-on-carbon (10%) catalyst (100 mg). The mixture is reduced under atmospheric pressure and room temperature for 20 minutes. Catalyst is then filtered and the filtrate is evaporated to dryness under reduced pressure. The two methylamino isomers are separated by partition column chromatography.

EXAMPLE 9 Preparation of 11a-chloro-5-a-hydroxy-6-methylene- 7- and/ or 9-nitrosotetracycline A solution of 50 mg. of 11a-chloro-5-u-hydroxytetracycline and 10 mg. of sodium nitrite in 5 m1. of hydrogen fluoride is stirred at ice-bath temperature for 45 minutes. The excess hydrogen fluoride is evaporated by passing over its surface a stream of nitrogen. The residue is dissolved in one ml. of methyl Cellosolve, diluted with ether and filtered.

mixture of Example 1 (100 mg.; 0.201

7 EXAMPLE 10 Preparation of 7- and/ or 9-amino-5-a-hydr0xy-6- methylenetetracycline The product from the previous example is dissolved in ml. of methyl Cellosolve and reduced with palladium on carbon (10%) and hydrogen at S.T.P. for 30 minutes. The catalyst is filtered and the filtrate evaporated to one ml. and diluted with ether.

EXAMPLE 11 Preparation of 7- and/or 9-dimethylamino-5-ahydr0xy-6-methylenetetracycline If: 2 IIUCHM Rn- V-CONH,

6H l) on i wherein R is selected from the group consisting of hydrogen, a-hydroxy and B-hydroxy, and R and R are each selected from the groupconsisting of hydrogen,

mono(lower alkyl)amino and di(lower alkyl)amino with the proviso that R, and R cannot both be hydrogen; and the non-toxic acid-addition salts thereof.

2. A compound according to claim 1 wherein R is hydrogen, R7 is dimethylamino and R is hydrogen.

3. A compound according to claim 1 wherein R is ot-hydroxy, R is dimethylamino and R is hydrogen.

4. A compound according to claim 1 wherein R a-hydroxy, R is isopropylamino and R is hydrogen.

5. A compound according to claim 1 wherein R fl-hydroxy, R, is dimethylamino and R is hydrogen.

6. A compound according to claim 1 wherein R oc-hYdl'OXY, R is hydrogen and R is methylamino.

7. A compound according to claim 1 wherein R hydrogen, R is hydrogen and R is methylamino.

8. A compound according to claim 1 wherein R hydrogen, R is diethylarnino and R is hydrogen.

9. A compound according to claim 1 wherein R is hydrogen, R is dimethylamino and R is hydrogen; said compound being in the form of its hydrochloride salt.

10. A compound according to claim 1 wherein R is u-hydroxy, R is dimethylamino and R is hydrogen; said compound being in the form of its hydrochloride salt.

References Cited UNITED STATES PATENTS NICHOLAS S. RIZZO, Primary Examiner. 

1. A COMPOUND SELECTED FROM THE GROUP CONSITING OF 6-METHYLENETETRACYCLINES OF THE FORMULA: 